Studies in an experimental animal model have established that Mycobacterium bovis BCG and Mycobacterium smegmatis cell walls attached to oil droplets cause established tumors to regress and eliminate regional lymph node metastases when injected intralesionally. Components derived from these cell walls are currently in use clinically, and they also have demonstrated efficacy in the treatment of malignant disease. Components of these cell walls that are effective in tumor regression have in part been identified, and include the macromolecular lipopolysaccharide-mucopeptide complex of the mycobacterial cell wall and a glycolipid constituent of the free lipid extract of the cell wall. Both of these components contain esterified mycolic acids, which have been found to be essential for effective therapy, but the structural features of the mycolic acids and the cell wall lipopolysaccharide-mucopeptide complex that are requisite for activity have not been identified. The overall objective of this proposed research is to identify the structural features of mycolic acids and the cell wall lipopolysaccharide-mucopeptide complex that are necessary for antitumor activity in order, hopefully, to provide more active and less toxic immunotherapeutic agents for use in te treatment of cancer. Isolation procedures developed in this laboratory have provided, for the first time, mycolic acids as structurally pure molecular entities, which makes possible a thorough examination of their structures and the structural basis for their activity. In addition, a general synthesis of mycolic acid analogs is being developed which will also provide pure components for biological testing. Purification of mycobacterial lipids and glycolipids will utilize primarily the technique of high performance liquid chromatography, and their characterization will utilize high resolution mass spectrometry, combined gas-liquid chromatography-mass spectrometry, and high resolution nuclear magnetic resonance spectroscopy. Characterization of the cell wall complex will utlize the above techniques combined with 13C NMR spectroscopy of solid samples.